Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication. Since the discovery that EVs are capable of functionally transferring biological information, the potential use of EVs as drug delivery vehicles has gained considerable scientific interest. EVs may have multiple advantages over currently available drug delivery vehicles. Here, we review and discuss development of EVs as drug delivery vehicles.

1. Extracellularvesiclesas drug deliverysystems
Guide:
Dr N. S. Ranpise
Presented By:
Vishakha Vikram Deshmukh
M Pharm, Sem- I (Pharmaceutics)
Roll no.-506
Sinhgad College of Pharmacy, Vadgaon (Bk), Pune-41

2. Extracellular Vesicles
• Phospholipid-bilayer - enclosed vesicles secreted from all cell types.
• Diameter of vehicles - 30 nm to 1 μm.
• Specialized functions.
• Occurring from cells.
• Have low side effects.
VISHAKHA VIKRAM DESHMUKH
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Ref.: Van Niel, G., d'Angelo, G. and Raposo, G., 2018. Shedding light on the cell biology of extracellular vesicles. Nature reviews
Molecular cell biology, 19(4), pp.213-228.
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3. VISHAKHA VIKRAM DESHMUKH
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• Present in – serum, urine, breast milk, CSF, saliva, amniotic fluids, bile, etc.
• Key role in intracellular signaling, waste management, and coagulation.
• Contain several types of function molecules – Proteins, mRNAs, and miRNAs.
• Potential tools for a Targeted Drug Delivery System.
Ref.: Kalra, H., Drummen, G.P. and Mathivanan, S., 2016. Focus on extracellular vesicles: Introducing the next small big
thing. International journal of molecular sciences, 17(2), p.170.
Cont..
3

4. History
• Cell-derived vesicles were discovered in 1940.
• Preliminary studies addressing the significance of thromboplastin protein (Chargaff and West,
1946).
• Subcellular fraction under electron microscopy showed small vesicles, originating from
platelets termed platelet dust (Wolf, 1967).
• Diameter 20 and 50 nm, a density between 1.020 and 1.025 g/ml. (Wolf, 1967).
• Fetal calf serum was also found to contain numerous microvesicles diameters from 30 to 60 nm.
• When vesicles were isolated from conditioned culture medium of sleep reticulocytes named
exosome.
VISHAKHA VIKRAM DESHMUKH
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Ref.: Van der Pol, E., Böing, A.N., Harrison, P., Sturk, A. and Nieuwland, R., 2012. Classification, functions, and
clinical relevance of extracellular vesicles. Pharmacological reviews, 64(3), pp.676-705. 4

5. Types of Extracellular Vesicles
 Based on Biogenesis Extracellular Vesicles (EVs)
1. Exosome
2. Microvesicles
3. Apoptic bodies
VISHAKHA VIKRAM DESHMUKH
15/02/2022
Ref.: Igami, K., Uchiumi, T., Ueda, S., Kamioka, K., Setoyama, D., Gotoh, K., Akimoto, M., Matsumoto, S. and Kang, D., 2020. Characterization and
function of medium and large extracellular vesicles from plasma and urine by surface antigens and Annexin V. PeerJ Analytical Chemistry, 2, p.e4. 5

6. Unique Selling Points of Extracellular Vesicles
 Endogenous cellular sorting & packaging -
i. Extracellular vesicles cargo loading is completely systemic process.
ii. Endogenous cellular machinery can be used to produce the desired cargo.
 Intrinsic ability to cross physical barriers -
i. Efficiently cross biological barriers.
ii. Tissue level, cellular level and intracellular level for the delivery of therapeutics.
 Safety profile -
i. Minimally reactive to the immune system.
ii. Used in Mesenchymal Stem Cell therapies.
VISHAKHA VIKRAM DESHMUKH
15/02/2022
Ref.: Elsharkasy, O.M., Nordin, J.Z., Hagey, D.W., de Jong, O.G., Schiffelers, R.M., Andaloussi, S.E. and Vader, P., 2020.
Extracellular vesicles as drug delivery systems: Why and how?. Advanced drug delivery reviews, 159, pp.332-343. 6

7. VISHAKHA VIKRAM DESHMUKH
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Figure- Unique features of extracellular vesicles
Ref.: Turturici, G., Tinnirello, R., Sconzo, G. and Geraci, F., 2014. Extracellular membrane vesicles as a mechanism of cell-to-
cell communication: advantages and disadvantages. American Journal of Physiology-Cell Physiology, 306(7), pp.C621-C633.
Cont..
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8. How do EVs work
 Cargo loading into Extracellular vesicles
 Functionalized Extracellular Vesicles for targeted delivery
 Upscaling, isolation, storage, and GMP Production
VISHAKHA VIKRAM DESHMUKH
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Ref.: Elsharkasy, O.M., Nordin, J.Z., Hagey, D.W., de Jong, O.G., Schiffelers, R.M., Andaloussi, S.E. and Vader, P., 2020.
Extracellular vesicles as drug delivery systems: Why and how?. Advanced drug delivery reviews, 159, pp.332-343 8

9. Cargo loading into Extracellular vesicles
Techniques employed for endogenous loading:-
i. Electroporation
ii. Simple incubation
iii. Sonication
iv. Extrusion
v. Freeze-thawing
Techniques employed for exogenous loading:-
i. Passive endogenous loading
ii. Active endogenous loading
Cargo loading
Endogenous loading
Exogenous loading
VISHAKHA VIKRAM DESHMUKH
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Ref.: Vader, P., Mol, E.A., Pasterkamp, G. and Schiffelers, R.M., 2016. Extracellular vesicles for drug
delivery. Advanced drug delivery reviews, 106, pp.148-156. 9

10. VISHAKHA VIKRAM DESHMUKH
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Herrmann, I.K., Wood, M.J.A. and Fuhrmann, G., 2021. Extracellular vesicles as a next-generation drug
delivery platform. Nature nanotechnology, 16(7), pp.748-759.
Production process of drug-loaded Extracellular
vesicles
Figure- Drug-loading in extracellular vesicles

11. • Loading efficiency and functional delivery platform & choice of extracellular
vesicles-associated molecules used for targeted loading.
depends upon
VISHAKHA VIKRAM DESHMUKH
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Figure- process of endogenous and exogenous loading
Ref.: Pascucci, L., Coccè, V., Bonomi, A., Ami, D., Ceccarelli, P., Ciusani, E., Viganò, L., Locatelli, A., Sisto, F., Doglia, S.M. and Parati, E., 2014. Paclitaxel is incorporated
by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: a new approach for drug delivery. Journal of controlled release, 192, pp.262-270. 11

12. Functionalized Extracellular Vesicles for targeted delivery
• Unique beneficial feature for Targeted drug delivery
• Intrinsic targeting properties(some extent)
Upscaling, Isolation, Storage, and GMP Production
• Upscaling - no change in phenotype during the cell culture process.
• Isolation – focus on purity
• Storage – focus on stability & shelf-life
• GMP production – avoid cross-contamination
VISHAKHA VIKRAM DESHMUKH
15/02/2022
Ref.: Murphy, D.E., de Jong, O.G., Brouwer, M., Wood, M.J., Lavieu, G., Schiffelers, R.M. and Vader, P., 2019. Extracellular vesicle-
based therapeutics: natural versus engineered targeting and trafficking. Experimental & molecular medicine, 51(3), pp.1-12. 12

13. Advantage
i. Natural
ii. Low immunogenicity
iii. Stable in biological fluids
iv. Translocation through physical barriers
v. Used for targeted drug delivery
vi. Delivery with or without modification
vii. Unidirectional targeting or active targeting by modification
viii. Suitable for multi-drug delivery
ix. High Bioavailability and biocompatibility
VISHAKHA VIKRAM DESHMUKH
15/02/2022
Ref.: Tang, T.T., Lv, L.L., Lan, H.Y. and Liu, B.C., 2019. Extracellular vesicles: opportunities and challenges for the
treatment of renal diseases. Frontiers in physiology, 10, p.226. 13

14. Limitations
i. Unclear biochemical composition of extracellular vesicles
ii. Poorly described production/uptake mechanism
iii. Understudied immune clearance
iv. Lacking of GMP standard
v. Difficult high scale & efficient production
vi. Difficult to package through renal barriers
vii. Lacking (pre)clinical evaluation
VISHAKHA VIKRAM DESHMUKH
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Ref.:Kumar, A., Zhou, L., Zhi, K., Raji, B., Pernell, S., Tadrous, E., Kodidela, S., Nookala, A., Kochat, H. and Kumar, S., 2021. Challenges in biomaterial-based drug
delivery approach for the treatment of neurodegenerative diseases: Opportunities for extracellular vesicles. International Journal of Molecular Sciences, 22(1), p.138. 14

15. VISHAKHA VIKRAM DESHMUKH
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Applications of Extracellular Vesicles in Drug
Delivery
Extracellular vesicles source Engineered content Application
Human embryonic kidney (HEK293)
cells
Surface modified with anti-EGFR peptides
(GE11) by cell engineering; loaded with
miRNA/siRNA by donor cell transfection with
synthetic oligonucleotides
Inhibition of breast cancer tumor growth
Porcine peripheral blood
None; EVs were administered in combination
with Montanide adjuvant
Vaccination against porcine reproductive
and respiratory syndrome virus
Human mesenchymal stem cells None Treatment of chronic myocardial ischemia
Autologous tumor cells Loaded with methotrexate through incubation
of the donor cells with the drug
Vaccination against advanced lung cancer
and malignant pleural effusion
Mouse neuroblastoma (Neuro2A) cells loaded with siRNA via EV incubation with
cholesterol-conjugated siRNA
Improved in vitro siRNA delivery
Ref.: de Jong, O.G., Kooijmans, S.A., Murphy, D.E., Jiang, L., Evers, M.J., Sluijter, J.P., Vader, P. and Schiffelers, R.M., 2019. Drug
delivery with extracellular vesicles: from imagination to innovation. Accounts of chemical research, 52(7), pp.1761-1770.

16. • Exosomes are natural carriers of biomacromolecules, which make them attractive candidates for
delivering therapeutic biomolecules.
• Helpful to solve the problem of targeting drug delivery and to develop an effective treatment plan for
the disease.
• Modification can be used to enhance the ability of exosomes to target cells or organs and improve the
efficiency of targeting delivery.
Conclusion
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References
• Van Niel, G., d'Angelo, G. and Raposo, G., 2018. Shedding light on the cell biology of extracellular
vesicles. Nature reviews Molecular cell biology, 19(4), pp.213-228.
• Kalra, H., Drummen, G.P. and Mathivanan, S., 2016. Focus on extracellular vesicles: Introducing the next
small big thing. International journal of molecular sciences, 17(2), p.170.
• Van der Pol, E., Böing, A.N., Harrison, P., Sturk, A. and Nieuwland, R., 2012. Classification, functions, and
clinical relevance of extracellular vesicles. Pharmacological reviews, 64(3), pp.676-705.
• Igami, K., Uchiumi, T., Ueda, S., Kamioka, K., Setoyama, D., Gotoh, K., Akimoto, M., Matsumoto, S. and
Kang, D., 2020. Characterization and function of medium and large extracellular vesicles from plasma and
urine by surface antigens and Annexin V. PeerJ Analytical Chemistry, 2, p.e4.
• Elsharkasy, O.M., Nordin, J.Z., Hagey, D.W., de Jong, O.G., Schiffelers, R.M., Andaloussi, S.E. and Vader, P.,
2020. Extracellular vesicles as drug delivery systems: Why and how?. Advanced drug delivery reviews, 159,
pp.332-343
17

18. VISHAKHA VIKRAM DESHMUKH
15/02/2022
• Turturici, G., Tinnirello, R., Sconzo, G. and Geraci, F., 2014. Extracellular membrane vesicles as a mechanism of cell-
to-cell communication: advantages and disadvantages. American Journal of Physiology-Cell Physiology, 306(7),
pp.C621-C633.
• Elsharkasy, O.M., Nordin, J.Z., Hagey, D.W., de Jong, O.G., Schiffelers, R.M., Andaloussi, S.E. and Vader, P., 2020.
Extracellular vesicles as drug delivery systems: Why and how?. Advanced drug delivery reviews, 159, pp.332-343.
• Vader, P., Mol, E.A., Pasterkamp, G. and Schiffelers, R.M., 2016. Extracellular vesicles for drug delivery. Advanced
drug delivery reviews, 106, pp.148-156.
• Pascucci, L., Coccè, V., Bonomi, A., Ami, D., Ceccarelli, P., Ciusani, E., Viganò, L., Locatelli, A., Sisto, F., Doglia,
S.M. and Parati, E., 2014. Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that
inhibit in vitro tumor growth: a new approach for drug delivery. Journal of controlled release, 192, pp.262-270.
• Murphy, D.E., de Jong, O.G., Brouwer, M., Wood, M.J., Lavieu, G., Schiffelers, R.M. and Vader, P., 2019.
Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking. Experimental &
molecular medicine, 51(3), pp.1-12
18

19. VISHAKHA VIKRAM DESHMUKH
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Thank You!
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